CN104364641A - Quantification of an analyte in serum and other biological matrices - Google Patents
Quantification of an analyte in serum and other biological matrices Download PDFInfo
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- CN104364641A CN104364641A CN201280053124.5A CN201280053124A CN104364641A CN 104364641 A CN104364641 A CN 104364641A CN 201280053124 A CN201280053124 A CN 201280053124A CN 104364641 A CN104364641 A CN 104364641A
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J49/00—Particle spectrometers or separator tubes
- H01J49/0027—Methods for using particle spectrometers
- H01J49/0031—Step by step routines describing the use of the apparatus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J49/00—Particle spectrometers or separator tubes
- H01J49/02—Details
- H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
- H01J49/0431—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components for liquid samples
-
- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J49/00—Particle spectrometers or separator tubes
- H01J49/02—Details
- H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
- H01J49/0431—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components for liquid samples
- H01J49/0445—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components for liquid samples with means for introducing as a spray, a jet or an aerosol
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J49/00—Particle spectrometers or separator tubes
- H01J49/02—Details
- H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
- H01J49/0459—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components for solid samples
- H01J49/0463—Desorption by laser or particle beam, followed by ionisation as a separate step
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J49/00—Particle spectrometers or separator tubes
- H01J49/02—Details
- H01J49/10—Ion sources; Ion guns
- H01J49/16—Ion sources; Ion guns using surface ionisation, e.g. field-, thermionic- or photo-emission
- H01J49/168—Ion sources; Ion guns using surface ionisation, e.g. field-, thermionic- or photo-emission field ionisation, e.g. corona discharge
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
- G01N27/622—Ion mobility spectrometry
- G01N27/624—Differential mobility spectrometry [DMS]; Field asymmetric-waveform ion mobility spectrometry [FAIMS]
Abstract
Methods and systems for quantifying analytes in a biological sample are provided comprising preparing a biological sample for mass spectrometric analysis, utilizing an ionization source to ionize at least a portion of the prepared biological sample to generate an ionized analyte flow, introducing the ionized analyte flow into a differential mobility spectrometer set at a compensation voltage selected to extract ionized analyte molecules from the ionized analyte flow, introducing an output analyte flow of the differential mobility spectrometer into a mass spectrometer to detect and quantify analyte ions in the output analyte flow.
Description
cROSS REFERENCE TO RELATED reference
Subject application request on October 26th, 2011 file an application the 61/551st, the right of priority of No. 489 U.S. Provisional Application cases, the mode that described U.S. Provisional Application case is quoted in full is incorporated herein, and subject application request on October 10th, 2012 file an application the 61/711st, the right of priority of No. 871 U.S. Provisional Application cases, the mode that described U.S. Provisional Application case is quoted in full is incorporated herein.
Technical field
The present invention relates generally to the method and system for using mass spectrum to detect and quantize the analysis thing in such as serum and other biological specimen equal samples.
Background technology
The Mass Spectrometer Method of analysis thing contained in complex matrices and quantification need high-resolution separation technology to reduce the effect of the material disturbed in sample usually.By way of example, most of mass spectrophotometry utilizes the selectivity that the downstream optical spectrum of liquid chromatography isolation technics analyte of interest to improve detects.Although this little isolation technics can produce quantize more accurately, both sample preparation and chromatographic resolution are all consuming time and cost is high, therefore reduce flux.
Therefore, need the flux with increase to allow the selective enumeration method of institute's analyte of interest (testosterone such as, in serum and vitamin D) in complex biological sample and the method and system of quantification simultaneously.
Summary of the invention
According to various embodiment, be provided for herein using mass spectrum to detect and/or the method and system of analysis thing in quantized samples.As described below, these method and system can realize detection and/or the quantification of the analysis thing in complex matrices when not using isolation technics consuming time.By way of example, the testosterone that laser desorption thermoionization can be utilized to quantize rapidly in multiple biological specimen (such as, serum) in conjunction with differential mobility spectroscopic methodology and the concentration of vitamin D.In various embodiments, analyze thing and can comprise 25-OH-vitamine D3.In various embodiments, analyze thing and can comprise 25-OH-calciferol.In various embodiments, ionic mobility spectrograph can comprise the FAIMS device of (for example) differential mobility spectrometer (DMS), various geometric configuration, such as parallel-plate, meander electrode or cylindrical FAIMS device and other.In many examples, this can perform when not using liquid chromatography isolation technics before ionization.
According to various embodiment, disclose a kind of method of the analysis thing quantized in biological specimen.Described method comprise for the preparation of mass spectrophotometry biological specimen and utilize ionization source to produce the ionization at least partially of described prepared biological specimen through ionization analyte stream.By described through ionization analyte stream be incorporated into be set in through select with from described through the differential mobility spectrometer of ionization analyte stream extraction under the bucking voltage of ionization analyte molecule, and being incorporated into described differential mobility spectrometer in mass spectrometer to detect and to quantize the described analyte ions through exporting in analyte stream through exporting analyte stream.In various embodiments, described analysis thing can comprise testosterone.In various embodiments, described analysis thing can comprise vitamin D.In various embodiments, described analysis thing can comprise 25-OH-vitamine D3.In various embodiments, described analysis thing can comprise 25-OH-calciferol.
In an aspect, ionization source can comprise laser diode thermal desorption (LDTD) ionization source.In certain embodiments, ionization source can comprise diode laser to give prepared biological specimen with biological specimen described in desorb at least partially by heat energy.In related fields, ionization source can comprise for will through the Ionized APCI ionizer of desorb biological specimen.
In all fields, mass spectrometer can comprise tandem mass spectrometer.For example, tandem mass spectrometer can be triple quadrupole mass spectrometer.In an aspect, described method can comprise at least one precursor-product ion of the m/z value with about 289/97,289/109 and 292/97 to transition.In an aspect, described method can comprise at least one precursor product ion of detecting and having a m/z value of about 401.3/365.2,401.3/257.1 and 401.3/201.1 to transition.In various embodiments, described method can comprise at least one precursor-product ion of detecting and having a m/z value of about 413.3/355.2 and 413.3/337.2 to transition.
Described differential mobility spectrometer can have various configuration.For example, bucking voltage can be about 4 volts.In this way, described differential mobility spectrometer can prioritised transmission testosterone (for example).In various embodiments, bucking voltage can in the scope of about 4 volts to about 12 volts.In all fields, bucking voltage can in the scope of about 4 volts to about 14 volts.
In certain aspects, biological specimen can be prepared when not needing liquid chromatography to be separated.In related fields, by performing liquid-liquid extraction and before ionization step, sample being prepared in biological specimen drying.In certain embodiments, described ionization source can rapidly by multiple sample ions.For example, by Ionized for the first biological specimen about 10 seconds, ionization source can be extra through ionization analyte stream to produce by the ionization at least partially of extra prepared biological specimen.In various embodiments, described biological specimen can be placed in the sample plane of described ionization source.For example, described biological specimen can be dry.
In certain aspects, biological specimen can be the one in blood, blood plasma, serum, oral fluid and urine.In certain embodiments, the quantitative restriction of about 0.1ng/mL analysis thing in biological specimen can be detected.In other words, method described herein can detect the analyte concentration being low to moderate 0.1ng/mL in biological specimen.In various embodiments, analyze thing and can comprise testosterone.In various embodiments, analyze thing and can comprise vitamin D.In various embodiments, analyze thing and can comprise 25-OH-vitamine D3.In various embodiments, analyze thing and can comprise 25-OH-calciferol.
In an aspect, a kind of mass spectrometer system is disclosed.Described system comprises ionization source, and described ionization source has and is configured to heat to be applied to sample to cause the thermal source of the desorb at least partially of described sample and to be used for the described ionization at least partially through desorption part to produce the ionizer through ionization sample.Described system also comprises the differential mobility spectrometer be communicated with described ionization source.Described differential mobility spectrometer can from described ionization source receive described through ionization sample at least partially and the ion of the selected differential mobility of transmission.The mass spectrometer be communicated with described differential mobility spectrometer is configured to the described ion analyzing the described selected differential mobility received from described differential mobility spectrometer.In various embodiments, described analysis thing can comprise testosterone.In various embodiments, described analysis thing can comprise vitamin D.In various embodiments, described analysis thing can comprise 25-OH-vitamine D3.In various embodiments, described analysis thing can comprise 25-OH-calciferol.
In certain embodiments, described thermal source can comprise laser diode.For example, described laser diode can be configured to produce the laser beam of the sample be directed to just containing described sample.
Described ionizer can have various being configured to and produce through ionization sample.In various embodiments, described ionizer can comprise atmospheric pressure chemical ionization source.In an aspect, described ionizer comprises the ionization pin producing corona discharge.In another aspect, described ionizer can comprise and is configured to produce for by the ultraviolet source of photoionization by the described light beam through desorb sample ions.In various embodiments, described mass spectrometer can comprise tandem mass spectrometer.In all fields, described mass spectrometer can be triple quadrupole mass spectrometer.In all fields, described system can detect at least one precursor-product ion of the m/z value with about 289/97,289/109 and 292/97 to transition.In all fields, described system can detect have about 401.3/365.2,401/257.1 and at least one precursor-product ion of m/z value of 401.3/201.1 to transition.In various embodiments, described system can detect at least one precursor-product ion of the m/z value with about 413.3/355.2 and 413.3/337.2 to transition.In various embodiments, described differential mobility spectrometer can have various configuration.For example, bucking voltage can be about 4 volts.In this way, described differential mobility spectrometer can prioritised transmission testosterone (for example).In various embodiments, bucking voltage can in the scope of about 4 volts to about 12 volts.In all fields, bucking voltage can in the scope of about 4 volts to about 14 volts.
Accompanying drawing explanation
Those skilled in the art will appreciate that, hereafter described graphic only for illustration of object.The scope of described graphic teachings of not intending to limit applicant by any way.
Fig. 1 illustrates the method for analyte quantification according to the various embodiments of the teachings of applicant.
Fig. 2 illustrates the system for analyte quantification according to the various embodiments of the teachings of applicant.
Fig. 3 shows the schematic diagram according to laser diode thermal desorption (LDTD) ionization source of the teachings of applicant.
Fig. 4 shows the reduction of the interference of various embodiments when series connection uses LDTD source and ion mobility device to analyze through blending serum testosterone caliberator in blank according to the teachings of applicant.
Fig. 5 displaying is according to the accuracy of the various embodiments of the teachings of applicant and repeatability.
Fig. 6 shows the typical curve result according to the various embodiments of the teachings of applicant.
Fig. 7 shows the typical curve result according to the various embodiments of the teachings of applicant.
Fig. 8 shows the reduction disturbed when connecting and using LDTD source and ion mobility device to analyze vitamin D according to the various embodiments of the teachings of applicant.
Fig. 9 shows according to the various embodiments of the teachings of applicant when only using LDTD source and turning off ion mobility device to analyze interference during vitamin D.
Figure 10 shows the comparison according to the interference when series connection use LDTD source and ion mobility device are to analyze vitamin D and when only using LDTD and turned off by ion mobility device in blank of the various embodiments of the teachings of applicant.
Figure 11 shows the typical curve result according to the various embodiments of the teachings of applicant.
Embodiment
Those skilled in the art will appreciate that, method and system described herein are non-restrictive illustrative embodiment and the scope of the disclosure of applicant is only defined by claims.Although describe the teachings of applicant in conjunction with various embodiment, do not intend the teachings of applicant to be defined in this little embodiment.On the contrary, the teachings of applicant contain variously to substitute, amendment and equivalents, as those skilled in the art will understand.Or the feature that describe illustrated in conjunction with one exemplary embodiment can with the Feature Combination of other embodiment.Amendment and change are intended to be included in the scope of the disclosure of applicant a bit for these.
According to various embodiment, be provided for the method for the institute's analyte of interest quantized in biological specimen.Fig. 1 describes the one exemplary embodiment being used for the method for analyte quantification according to the teachings of applicant.As demonstrated, described method can comprise and prepare biological specimen, by sample ions, be input in differential mobility spectrometer (DMS) and the output of quality analysis DMS by ion.
As those skilled in the art will understand, can prepare containing maybe containing the sample of analyte of interest to some extent for mass spectrophotometry.By way of example, sample can be biological specimen, such as blood, serum, blood plasma, oral fluid or urine.Known various technology in the technique according to teachings amendment herein can be used to prepare sample.In certain embodiments, sample preparation steps consuming time can be eliminated according to the method for the teachings of applicant.For example, in certain embodiments, prepare sample not need to comprise use liquid chromatography with the possible interfering material existed in separating sample.In an aspect, sample preparation can comprise execution liquid-liquid extraction and drying, as discussed herein.
Can then by prepared sample ions.Various ionization techniques known in the technique according to teachings amendment herein can be used prepared sample ions.In one embodiment, laser diode thermal desorption can be used the sequentially ionization of multiple sample, as discussed herein.In an exemplary embodiment, prepared sample in sample plane is placed in such as by carrying out desorb to sample plane (such as, with sample thermo-contact a part) or sample own radiation to make a part for sample vaporize.Sample can be transferred to ionizer with will through desorb sample ions through desorption part.As those skilled in the art will understand, the various ionization techniques revised according to teachings herein can be utilized sample ions.By way of example, atmospheric pressure chemical ionization or photoionization can be used a part of ionization through desorb sample.
After ionization, a part for sample can be delivered to differential mobility spectrometer with selected through ionization analysis thing from extracting at least in part through ionization sample.As those skilled in the art will understand, the bucking voltage of differential mobility spectrometer can through selecting with prioritised transmission institute analyte of interest.By way of example, bucking voltage can be set as that about 4 volts with will through ionization testosterone prioritised transmission to downstream quality analyser.In various embodiments, bucking voltage can in the scope of about 4 volts to about 12 volts.In all fields, bucking voltage can in the scope of about 4 volts to about 14 volts.
The ion exiting DMS can such as, by downstream spectrometer analysis, tandem mass spectrometer.As those skilled in the art will understand, the various technology for analyzing the ion transmitted by differential mobility spectrometer can be utilized.By way of example, observable select MRM transition to find analyte of interest to realize the quantitative of analysis thing in sample.Detect wherein and quantize in an one exemplary embodiment of testosterone, at least one product-precursor ions of the m/z value with about 289/97,289/109 and 292/97 can be used transition.Detect wherein and quantize in an one exemplary embodiment of 25-OH-vitamine D3, at least one product-precursor ions of the m/z value with about 401.3/365.2,401.3/257.1 and 401.3/201.1 can be used transition.Detect wherein and quantize in an one exemplary embodiment of 25-OH-calciferol, at least one product-precursor ions of the m/z value with about 413.3/355.2 and 413.3/337.2 can be used transition.
According to various embodiment, be provided for the mass spectrometer system of the institute's analyte of interest quantized in biological specimen.Fig. 2 describes an one exemplary embodiment of the system being used for analyte quantification.As demonstrated, described system comprises the ionization source and downstream quality analyser that are operatively coupled to differential mobility spectrometer (DMS).As those skilled in the art will understand, can be used for quantizing various analysis things existing in various biological specimen, such as testosterone and vitamin D according to the system of the teachings of applicant.In various embodiments, analyze thing and can comprise 25-OH-vitamine D3.In various embodiments, described analysis thing can comprise 25-OH-calciferol.
As discussed herein, sample can be prepared before ionization.Can then use various ionization source by sample ions.For example, APCI and photoionization can be used sample ions.In one embodiment, ionization source can be laser diode thermal desorption ionization source, and it can sample prepared by desorb and/or ionization effectively.By Fitow Neose tech. Inc. (Phytronix Technologies of Quebec, CAN, Inc.) the LDTD S-960/S-3480 manufactured is can (for example, as described in following instance part) example of this provenance of operating as discussed.
Can then by through ionization sample delivery to DMS, institute's analyte of interest (such as, testosterone, vitamin D etc.) is optionally transferred to downstream quality analyser by it.In various embodiments, chemical modification agent can be introduced in systems in which, for example, in DMS interface.Although DMS and mass-synchrometer are depicted as independent assembly, be understood by those skilled in the art that DMS can be embodied as integrated unit.By way of example, SelexION
tMion mobility device can with as Massachusetts not thunder Framingham (Framingham, MA) AB SCIEX produce
5500 triple quadrupole mass spectrometers are directly situated between and connect.As those skilled in the art will understand, mass-synchrometer can have various configuration and can be the independent mass analyser being operatively coupled to DMS.By way of example, mass-synchrometer can be tandem mass spectrometer, triple quadrupole, QqTOF or trap TOF mass spectrometer.
Example
The teachings of applicant can with reference to hereafter presented example and the data obtained even comprehend.One of ordinary skill in the art are by considering this instructions and putting into practice this teachings disclosed herein by other embodiment of the teachings of apparent applicant.These examples are intended only to be considered as exemplary.
Laser Diode Thermal Desorption
tM(LDTD) ionization source technology is coupled to and is equipped with SelexION
tMthe mass spectrometer of difference ion mobility device, thus the high flux capacity realizing the analysis of the analysis thing be used in bio-matrix.LDTD source allows sample fast laser thermal desorption under atmospheric pressure, the ionization of follow-up then APCI type.The blank interference-limited that the lower limit of quantitation that the LDTD-MS/MS analyzing thing analyzes can produce by the existence due to homogeneity dystopy analysis thing, but, the SelexION between LDTD source and tandem mass spectrometer
tMthe interpolation of difference ion mobility device provides the selectivity of enhancing, and effectively allows homogeneity dystopy to analyze the separation of thing.Fig. 3 shows the schematic diagram of LDTD ionization source.LDTD uses laser diode produce and control the heat in sample support, and described sample support is 96 orifice plates.Then shift energy to make sample dry via sample holder, sample was vaporized before by the gas delivery in APCI district.Owing to there is not solvent and mobile phase, there is the protonated sign ionization of high-level efficiency to the strong opposing that ion suppresses.This allows the high throughput of 7 seconds sample-sample analysis time (without postponing).Use differential mobility spectroscopic methodology, SelexION
tMion mobility device realizes being better than the selectivity of the enhancing that traditional MS/MS analyzes by providing the orthogonal means of separation homogeneity dystopy material.When not using liquid chromatography to be separated homogeneity dystopy material before being analyzed by MS/MS, this ability becomes and is even more important.In the illustration being described, ion mobility unit is directly situated between and receives
5500 mass spectrometric front portions, and by by bucking voltage (CoV) parameter be tuned to the value of 4V and the detection optimized for testosterone.Sample is prepared as follows:
1. the serum sample of 100 μ L is transferred to borosilicate test tube.
2. add the IS working stock solution (10ng/mL) of 20 μ L to each test tube.
3. the NaOH (IN) of 300 μ L to be drawn in borosilicate test tube and to use vortex mixer to mix 10 seconds.
4. add the MTBE of 400 μ L to test tube and use vortex mixer to mix 30 seconds.
5. at ambient temperature test tube is vertically placed 5 minutes and with 14000rpm centrifugal 5 minutes.
6. the upper layer clear liquid of 200 μ L is transferred to clean borosilicate test tube and at 45 DEG C in N2 gas evaporation drying.
7. sample restores and uses vortex mixer to mix 60 seconds in 40 μ L reconstituted solution.
8. then with 14000rpm by centrifugal for test tube 1 minute to precipitate any insoluble or particle matter.
9. clean deposit-free solution is transferred to the borosilicate HPLC bottle being equipped with 200 μ L interpolation pipes.
As demonstrated in Figure 4, when series connection uses LDTD source and SelexION
tMion mobility device with analyze through fusion serum testosterone caliberator time, the interference in blank is reduced to close to zero from 65%, thus produce lower limit of quantitation 10x improve.Sample preparation is formed than using the simple liquid-liquid extraction of ethyl acetate to human plasma by with 1:4v/v.The upper layer of 2 μ l is directly stored in 96 orifice plates and allows dry before analysis.These results represent and are better than there is not SelexION
tMthe remarkable improvement of the result obtained when ion mobility device.
Lower limit of quantitation is 0.1ng/ml, and linearly fabulous more than 5 orders of magnitude of chemical examination display, wherein r2=0.99972.Accuracy and repeatability in acceptance value, as demonstrated in Figure 5.Sample-sample is only 7 seconds working time.By contrast, the equivalent analysis of conventional LC-MS/MS is used usually will to need about 3 to 5 minutes by every sample.
Result shows that baseline significantly reduces and is reduced to the point that may realize suitable LOQ, as demonstrated in Figure 4 in the extraction of L/L serum.This realizes when not introducing the chemical modification agent in DMS interface.Under these conditions, testosterone has the best COV of 4V.Additional selective may be realized by introducing improver.
Use LDTD/SelexION
tMcombination finds and analyze extract through admixing serum calibration agent.Repeat described experiment and obtain two standard sets Dependence Results, as in Fig. 6 and 7 show.Described result shows good linear.
Series connection uses LDTD source and ion mobility device (being Selexlon in this example) to analyze vitamin D to be separated the homogeneity dystopy interference of 7-Alpha-hydroxy-4-cholestene-3-ketone.Laser Diode Thermal Desorption
tM(LDTD) ionization source is coupled to and is equipped with SelexION
tMthe mass spectrometer of difference ion mobility unit, thus the high flux capacity of the analysis of the 25-OH Vintamin D2 realizing being used in bio-matrix and D3, wherein sample-sample analysis time is 9 seconds.The preparation of plasma sample adds the follow-up liquid-liquid extraction of hexane that then uses of methyl alcohol by protein precipitation to form.The upper layer of 5 μ l to be left in proprietary 96 orifice plates and to allow dry before analysis.To be separated by raw homogeneity dystopy compound 7 Alpha-hydroxies-cholestene-3-ketone in the MS/MS transition of known disturbances 25-OH-D3 by the effect of differential mobility spectrometer, as individual criterion specific desorb demonstrated.Use and prepare calibration curve according to the multistage caliberator of Chromsystem setting.By preparing extra curve level with through stripping serum-dilution caliberator.Carry out multiple test for checking.By first measuring the original level of 6 different plasma samples and matrix effect being evaluated in the 25-OH-D3 fusion of itself and known quantity.Observe constant interior life+through fusion quantity.Specificity is measured as comparing of under quantitative restriction blank level peak region and signal area.Repeatable test is for in the daytime and the measurement repeated between the some other day.In matrix, evaluate stability, extract at room temperature and 4 DEG C and be dried on Lazwell plate.Finally, in QC fusion 6 kinds of concomitant drugs (caffeine, acetaminophen etc.) to verify possible interference.Scope from 1 to 65ng/ml and 1.5 to 94ng/ml be quantitatively respectively used to 25-OH-vitamine D3 and D2.For two kinds of compounds, blank level is less than 20% of LOQ.For assessment accuracy and accuracy, in triplicate calibration point and QC are analyzed.The repeatable scope of n=3 is from 0.6% to 12.3%.Institute's calculating concentration of QC is in 15% of institute's report value.Relation between LC MS/MS and LDTD MS/MS sample is by r
2=0.952 expresses.Meet the guilding principle of the supervision environment of all certificate parameters at 9 seconds inner analysis samples with LDTD MS/MS.
Sample is prepared as follows:
According to the plasma standard of Chromsystem
Hang oneself the blank of dual stripping blood plasma: concentration <1ng/ml
The serial dilution of blood plasma
The internal standard of deuterate
The blood plasma of 20 μ l is pulverized together with the MeOH of 40 μ l
The water saturation NaCl+40 μ l hexane of supernatant+20 μ l of 40 μ l
2 μ l sediments
As demonstrated in Figure 8, as LDTD source and ion mobility device and differential mobility device SelexION
tM(in this example) connect and series connection use to analyze vitamin D time, the interference from 7-Alpha-hydroxy-4-cholestene-3-ketone is 0.6%.By contrast, as demonstrated in Figure 9, when only using LDTD source and differential mobility device turns off, the interference from 7-Alpha-hydroxy-4-cholestene-3-ketone is 16.9%.
Figure 10 shows as LDTD source and SelexION
tMwhen ion mobility device is connected and connects for analyzing vitamin D, the interference reduction compared with when differential mobility device turns off when only using LDTD source in blank.
Figure 11 shows the typical curve result of good linear.
Based on embodiment as described above, be understood by those skilled in the art that further feature and the advantage of the method and system of the teachings according to applicant.Therefore, the teachings of applicant is not by the content constraints of particular display and description, unless appended claims instruction.The mode that all publication quoted herein and list of references are quoted all in full is clearly incorporated herein.
Claims (20)
1. quantize a method for the analysis thing in biological specimen, it comprises:
Prepare biological specimen for mass spectrophotometry,
Utilize ionization source by the ionization at least partially of described prepared biological specimen to produce through ionization analyte stream,
By described through ionization analyte stream be incorporated into be set in through select with from described in the differential mobility spectrometer of ionization analyte stream extraction under the bucking voltage of ionization analyte molecule,
The output analyte stream of described differential mobility spectrometer is incorporated in mass spectrometer with the analyte ions detected and quantize in described output analyte stream.
2. method according to claim 1, wherein said analysis thing comprises testosterone.
3. method according to claim 1, wherein said analysis thing is selected from the group be made up of 25-OH-calciferol and 25-OH-vitamine D3.
4. method according to claim 1, wherein said ionization source comprises laser diode thermal desorption LDTD ionization source.
5. method according to claim 1, wherein said mass spectrometer comprises tandem mass spectrometer.
6. method according to claim 5, wherein detect have about 289/97,289/109,292/97, at least one precursor-product ion of the m/z value of 401.3/365.2,401.3/257.1,401.3/201.1,413.3/355.2 and 413.3/337.2 is to transition.
7. method according to claim 1, wherein said bucking voltage is about 4 volts to about 14 volts.
8. method according to claim 1, wherein detects described testosterone with the quantitative restriction of about 0.1ng/mL.
9. method according to claim 1, wherein said ionization source comprises diode laser heat energy is given described prepared biological specimen with biological specimen prepared described in desorb at least partially.
10. method according to claim 9, wherein said ionization source comprise for by described through the Ionized APCI ionizer of desorb biological specimen.
11. methods according to claim 1, wherein prepare described biological specimen when being separated without the need to liquid chromatography.
12. methods according to claim 11, wherein prepare described sample packages containing performing liquid-liquid extraction and before described ionization step, described biological specimen is dry.
13. methods according to claim 12, it comprises further and utilizes described ionization source by the ionization at least partially of extra prepared biological specimen with being produced additionally through ionization analyte stream in Ionized about 10 seconds by described prepared biological specimen.
14. 1 kinds of mass spectrometer systems, it comprises:
Ionization source, its have be configured to heat to be applied to sample with produce through desorb sample thermal source and be used for by the described ionization at least partially through desorb sample to produce the ionizer through ionization sample,
Differential mobility spectrometer, it is communicated with described ionization source, described differential mobility spectrometer be configured to from described ionization source receive described through ionization sample at least partially and the ion of the selected differential mobility of transmission, and
Mass spectrometer, it is communicated with described differential mobility spectrometer, and described mass spectrometer is configured to the described ion analyzing the described selected differential mobility received from described differential mobility spectrometer.
15. systems according to claim 14, wherein said sample packages is containing testosterone.
16. systems according to claim 14, wherein said sample is selected from the group be made up of 25-OH-calciferol and 25-OH-vitamine D3.
17. systems according to claim 14, wherein said thermal source comprises laser diode.
18. systems according to claim 17, wherein said ionizer comprises atmospheric pressure chemical ionization source.
19. systems according to claim 14, wherein said ionizer comprises and is configured to produce for by the ultraviolet source of photoionization by the described light beam through desorb sample ions.
20. systems according to claim 14, wherein said mass spectrometer comprises tandem mass spectrometer.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201161551489P | 2011-10-26 | 2011-10-26 | |
| US61/551,489 | 2011-10-26 | ||
| US201261711871P | 2012-10-10 | 2012-10-10 | |
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| US9970900B2 (en) * | 2014-08-05 | 2018-05-15 | Dh Technologies Development Pte. Ltd. | Methods for distinguishing dioleinates of aged and non-aged olive oil |
| US10309930B2 (en) | 2014-10-08 | 2019-06-04 | Dh Technologies Development Pte. Ltd. | Dynamic orthogonal analysis method |
| CN106248773B (en) * | 2016-07-19 | 2018-11-23 | 中国检验检疫科学研究院 | It is a kind of quickly to measure vitamin B in multidimensional piece1With ascorbic method |
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| EP2776822B1 (en) | 2016-09-21 |
| CA2867996A1 (en) | 2013-05-02 |
| EP2776822A1 (en) | 2014-09-17 |
| EP2776822A4 (en) | 2015-05-27 |
| WO2013061146A1 (en) | 2013-05-02 |
| US20140291505A1 (en) | 2014-10-02 |
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| US9209003B2 (en) | 2015-12-08 |
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